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理性设计大幅提高一种丝孢堆黑粉菌来源脂肪酶催化活性——用于合成莫西沙星手性中间体
Xue Cai, Jiang-Wei Shen, Yu Qiang, Jing Hua, Zhang-Qi Ma, Zhi-Qiang Liu, Yu-Guo Zheng
工程（英文） ›› 2022, Vol. 19 ›› Issue (12) : 207-216.
PDF(2590 KB)
PDF(2590 KB)
理性设计大幅提高一种丝孢堆黑粉菌来源脂肪酶催化活性——用于合成莫西沙星手性中间体
Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design
通过脂肪酶不对称拆分外消旋N-乙酰基-哌啶-2,3-二甲酸二甲酯[cis-(±)-1]，从而获得手性中间体 (S,S)-2,8-二氮杂双环壬烷，是合成氟喹诺酮类抗生素莫西沙星重要手性中间体的一条具有吸引力的工艺路线。在前期研究中，筛选得到了一株丝孢堆黑粉菌（SRL）来源的脂肪酶，该脂肪酶具有高热稳定性和pH值稳定性。但SRL来源的脂肪酶活性较低，无法满足工业化应用的需求。因此本研究依据前期南极假丝酵母来源脂肪酶B（CALB）改造策略，采用理性设计方法对SRL进行定点突变改造，获得一个双突变体SRLI194N/V195L。随后，确定了位于活性口袋外的环6 上两个关键氨基酸残基L145 和L154；获得四位点突变体SRL-I194N/V195L/L145V/L154G（V13），活性显著提高，达到87.8 U·mg−1，是野生型SRL的2195 倍（E > 200）。该突变体在50 ℃下的半衰期达到92.5 h。突变体V13（100 mg·L−1）能够高效拆分1 mol·L−1cis-(±)-1，2 h 内底物转化率达到49.9%，实现严格立体选择性（E > 200）。总体而言，研究发现了一株对cis-(±)-1 (S,S)-2,8-二氮杂双环壬烷具有高催化活性、严格立体选择性的脂肪酶，可应用于工业化生产，并为其他具有相似结构的脂肪酶和其他种类酶的活性提高提供了一种通用策略。
Lipase-catalyzed stereoselective resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate (cis-(±)-1) is an attractive route for the synthesis of (S,S)-2,8-diazobicyclo[4.3.0]nonane, an important chiral intermediate of the fluoroquinolone antibiotic, moxifloxacin. In our previous study, a lipase from Sporisorium reilianum (SRL) was identified to possess excellent thermostability and pH stability. However, the low enzymatic activity of the SRL is a challenge that must be addressed. A rational design was initially employed for SRL tailoring according to the engineered Candida antarctica lipase B (CALB), resulting in a beneficial variant called SRL-I194N/V195L. Subsequently, two key amino acid residues in loop 6, L145 and L154, which might modulate the lid conformation between open and closed, were identified. A tetra-site variant, SRL-I194N/V195L/L145V/L154G (V13), with a significantly enhanced activity of 87.8 U∙mg−1 was obtained; this value was 2195-fold higher than that of wild-type SRL. Variant V13 was used to prepare optically pure (2S,3R)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ((2S,3R)-1), resolving 1 mol∙L−1 cis-(±)-1 with a conversion of 49.9% in 2 h and absolute stereoselectivity (E > 200). Excellent stability with a half-life of 92.5 h was also observed at 50 °C. Overall, the study findings reveal a lipase with high activity toward cis-(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.
脂肪酶 / 丝孢堆黑粉菌 / 定点突变 / 分子动力学模拟 / 理性设计 / 莫西沙星
Lipase / Sporisorium reilianum / Site-directed mutagenesis / Molecular dynamics simulation / Rational design / Moxifloxacin
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